Influence of hypertension on serum concentration of type IV collagen antigens in streptozotocin-diabetic and non-diabetic rats

Summary The serum concentration of 7S collagen was measured radioimmunologically as a marker of basement membrane type IV collagen synthesis in diabetic and nondiabetic rats with Goldblatt hypertension. In non-diabetic rats the 7S collagen level was significantly raised after induction of hypertension (51%; p<0.001), and showed a positive correlation with relative heart weight as an integral parameter of hypertension (r= 0.63; p<0.01). In diabetic rats, which displayed a 7S collagen concentration roughly 2.5 times as high as the metabolically normal animals, the 7S collagen level was 27% higher in the hypertensive animals (p<0.01). There was no correlation with blood pressure or heart weight, but only a positive correlation with blood glucose (r=0.51; p<0.05). The results indicate that haemodynamic alterations may alter basement membrane collagen metabolism. However, type IV collagen metabolism in diabetes is influenced to a greater extent by metabolic than by haemodynamic factors.     

Type IV collagen antigens in serum of diabetic rats: a marker for basement membrane collagen biosynthesis

Summary The nature of type IV collagen antigens in the serum of streptozotocin diabetic rats was studied using radioimmunoassays for the N-terminal (7S-collagen) and C-terminal domain of type IV collagen. Type IV collagen antigen crossreacting with antibodies to the C-terminal domain was elevated from 32.0±5.36 ng/ml (n=10) in serum of normal rats to 94.9±24.5 ng/ml (n=10,P<0.0001) in serum of streptozotocin diabetic rats and could be normalized to 40.1±8.30 ng/ml (n=18) by insulin treatment. Molecular sieve chromatography of serum demonstrated a high molecular weight fraction containing the C-terminal and N-terminal domains and smaller material containing only the N-terminal domain. Degradation of the high molecular weight material by collagenase indicates that it consists of intact collagen type IV. Its relative proportion increased from 42% to 54% 4 weeks after diabetes induction. Together with unaltered clearance rates of 7S collagen in normal and diabetic rats, the data suggest that the increase of collagen type IV antigens in diabetic states reflects increased synthesis of collagen type IV.     

Glomerular structural and functional changes in a high-fat diet mouse model of early-stage Type 2 diabetes

Aims/hypothesis Type 2 diabetes often results in diabetic nephropathy, which is preceded by an elevated glomerular filtration rate (GFR). This study was designed to develop a mouse model of Type 2 diabetes and to elucidate the glomerular events in the early stages of diabetic nephropathy.Methods Four-week-old mice were fed a normal or high-fat (42% of total calories from fat) diet, and body weight, blood glucose, insulin, leptin, lipids and GFR were monitored from 9 to 21 weeks or longer after the feeding programme. Mesangial cell dedifferentiation was accessed by alpha-smooth muscle actin staining. Glomerular hypertrophy was determined using image analysis with haematoxylin–eosin staining. Matrix deposition was determined by type IV collagen staining.Results After 9 weeks, mice fed a high-fat diet weighed more than mice fed a normal diet (30.5±1.2 vs 22.3±0.5 g, p<0.05), and mice fed a high-fat diet were hyperinsulinaemic (283.9±69.7 vs 102.9±36.4 pmol/l, p<0.05), hyperglycaemic (8.0±0.6 vs 6.5±0.2 mmol/l, p<0.05) and their leptin levels were increased six-fold (1.48±0.45 vs 0.25±0.03 ng/ml, p<0.05). After 13 weeks, mice fed a high-fat diet showed hyperfiltration (GFR; 440±60 vs 210±10 µl/min, p<0.05). During the early stages of diabetic nephropathy, mesangial cell dedifferentiation was evident, shown by increased expression of alpha-smooth muscle actin in the glomeruli. After 9 weeks, mice fed a high-fat diet already demonstrated increased type IV collagen deposition. After 13 weeks, they developed enlarged glomerular tufts compared with those of their age-matched controls.Conclusions/interpretation The results of this study suggest that collagen IV deposition precedes the hyperfiltration and enlargement of glomeruli in early-stage diabetic nephropathy. Dedifferentiation of mesangial cells may be associated with collagen IV deposition.     

Serum 7S domain of type IV collagen levels in essential hypertension and hypertensive type 2 diabetic patients

Metabolic alteration of Type IV collagen occurs in micro- or macrovascular basement membrane of diabetic patients. Hypertension, a risk factor for clinical progression of diabetic vascular disease, may influence this metabolic alteration. The object of this study was to evaluate the serum 7S domain of type IV collagen (7S-collagen) levels in patients with essential hypertension and in Type 2 diabetic patients with or without hypertension and to investigate the relationship between the type IV collagen metabolism and the arterial blood pressure. Serum 7S-collagen levels in 18 patients with essential hypertension were significantly higher than in 24 normal subjects (4.2 ± 0.5 vs 3.6 ± 0.4 ng ml⁻¹ p < 0.01). Serum 7S-collagen levels in 28 normotensive diabetic patients (4.2 ± 0.5 ng ml⁻¹) were significantly higher than in normal subjects (p < 0.01). The serum 7S-collagen levels were significantly higher in 22 diabetic patients with hypertension (4.8 ± 0.6 ng ml⁻¹) than in the other groups. There was a significant correlation between the serum 7S-collagen levels and the systolic blood pressure in cases with essential hypertension (r = 0.59, p < 0.001) and in all diabetic patients (r = 0.52, p < 0.001), suggesting that elevation of the systolic blood pressure may influence the type IV collagen metabolism of vascular basement membrane. We conclude that the metabolic alteration of basement membrane occurring in patients with diabetes mellitus may worsen in the presence of high systolic blood pressure. © 1997 by John Wiley & Sons, Ltd.     

Glucose enhances type IV collagen production in cultured rat glomerular mesangial cells

Summary Type IV collagen production by cultured glomerular mesangial cells and the effect of glucose on it were evaluated in order to explore the possible contribution of mesangial cells to the accumulation of type IV collagen in mesangial matrix typically seen in diabetes. Type IV collagen was measured quantitatively by enzyme-linked immunosorbent assay. The majority of type IV collagen was secreted into culture media and secreted-type IV collagen increased with cell growth in early log phase and decreased in late log phase and after confluency. By exposing the cells to high concentrations of glucose (27.8 mmol/l), both secreted- and cell-associated-type IV collagens increased significantly compared with the cells cultured under normal glucose concentrations (5.6 mmol/l) or under equivalent concentrations of mannitol, resulting in a significant increase in total type IV collagen accumulation from 32.1±6.4 (under 5.6 mmol/l glucose) to 51.0±4.6 g/dish (mean ± SD, n=4) on day 4, from 113.6±6.6 to 156.8±7.1 on day 6, from 248.5±15.2 to 310.0±12.6 on day 8 and from 372.4±14.8 to 507.9±17.2 on day 12. These results indicate the importance of glucose-induced alteration of mesangial cell function in the development of diabetic mesangial expansion.     

Selective proteinuria in diabetic nephropathy in the rat is associated with a relative decrease in glomerular basement membrane heparan sulphate

Summary In the present study we investigated whether glomerular hyperfiltration and albuminuria in streptozotocin-induced diabetic nephropathy in male Wistar-Münich rats are associated with changes in the heparan sulphate content of the glomerular basement membrane. Rats with a diabetes mellitus duration of 8 months, treated with low doses of insulin, showed a significant increase in glomerular filtration rate (p<0.01) and effective renal plasma flow (p<0.05), without alterations in filtration fraction or mean arterial blood pressure. Diabetic rats developed progressive albuminuria (at 7 months, diabetic rats (D): 42±13 vs control rats (C): 0.5±0.2 mg/ 24 h, p<0.002) and a decrease of the selectivity index (clearance IgG/clearance albumin) of the proteinuria (at 7 months, D: 0.20±0.04 vs C: 0.39±0.17, p<0.05), suggesting loss of glomerular basement membrane charge. Light- and electron microscopy demonstrated a moderate increase of mesangial matrix and thickening of the glomerular basement membrane in the diabetic rats. Immunohistochemically an increase of laminin, collagen III and IV staining was observed in the mesangium and in the glomerular basement membrane, without alterations in glomerular basement membrane staining of heparan sulphate proteoglycan core protein or heparan sulphate. Giomerular basement membrane heparan sulphate content, quantitated in individual glomerular extracts by a new inhibition ELISA using a specific anti-glomerular basement membrane heparan sulphate monoclonal antibody (JM403), was not altered (median (range) D: 314 (152–941) vs C: 262 (244–467) ng heparan sulphate/mg glomerulus). However, the amount of glomerular 4-hydroxyproline, as a measure for collagen content, was significantly increased (D: 1665 (712–2014) vs C: 672 (515–1208) ng/mg glomerulus, p<0.01). Consequently, a significant decrease of the heparan sulphate/4-hydroxyproline ratio (D: 0.21 (0.14–1.16) vs C: 0.39 (0.30–0.47), p<0.05) was found. In summary, we demonstrate that in streptozotocin-diabetic rats glomerular hyperfiltration and a progressive, selective proteinuria are associated with a relative decrease of glomerular basement membrane heparan sulphate. Functionally, a diminished heparan sulphate-associated charge density within the glomerular basement membrane might explain the selective proteinuria in the diabetic rats.Abbreviations BW Body weight - ERPF effective renal plasma flow - GAG glycosaminoglycan - GBM glomerular basement membrane - GFR glomerular filtration rate - HS heparan sulphate - HSPG heparan sulphate proteoglycan - IDDM insulin-dependent diabetes mellitus - STZ streptozotocin     

Serum and urinary concentrations of type IV collagen and laminin as a marker of microangiopathy in diabetes.

Serum and urinary concentrations of type IV collagen and laminin were measured by enzyme-linked immunosorbent assay (ELISA) in diabetic patients and compared with normal control subjects. In diabetic patients with proteinuria or with renal insufficiency, serum and urinary concentrations of type IV collagen were higher than those of control subjects (p less than 0.005). Furthermore urinary concentrations of type IV collagen and laminin were significantly higher in diabetes, even in the absence of nephropathy, than in normal controls (p less than 0.05). Urinary concentrations of type IV collagen in patients with diabetes and microalbuminuria (0.73 +/- 0.11 mg mmol-1) were significantly higher than in diabetic patients without nephropathy (0.40 +/- 0.060 mg mmol-1) (p less than 0.025). Urinary concentrations of type IV collagen may have a role as an indicator of early diabetic nephropathy. Serum concentrations of type IV collagen in diabetic patients with retinopathy were significantly higher than in normal controls (p less than 0.025). However, urinary concentrations of type IV collagen (p less than 0.05) and serum concentrations of laminin (p less than 0.025) were significantly higher in diabetic patients than normal controls and the difference between patients with and without retinopathy was not significant.     

Decreased matrix degradation in diabetic nephropathy: effects of ACE inhibition on the expression and activities of matrix metalloproteinases

Aims/hypothesis: Extracellular matrix accumulation is thought to be involved in the pathogenesis of diabetic nephropathy. Increased matrix synthesis has been well documented but the effects of diabetes on degradative pathways, particularly in the in vivo setting, have not been fully explored. Furthermore, the effect of renoprotective therapies on matrix accumulation through these pathways has not been examined. We investigated the degradative pathway of type IV collagen and the effects of ACE inhibition in experimental diabetic nephropathy. Methods: Diabetes was induced in 16 rats by administrating streptozocin; 8 of the diabetic rats were allocated at random to receive the ACE inhibitor perindopril (2 mg/l) in their drinking water and 8 age and weight matched rats served as controls. Gene expression of matrix metalloproteinase (MMP) and tissue inhibitor of metalloproteinase (TIMP) was measured by RT-PCR and type IV collagen content by immunohistochemistry. MMP activities were determined by degradation of a radiolabelled substrate and by zymography. Results: Six months of diabetes was associated with a decrease in mRNA and enzymatic activity of MMP-9 (21 % and 51 % respectively, p < 0.05 vs control) and a 51 % increase in TIMP-1 mRNA (p < 0.05 vs control). By contrast, MMP-2 mRNA was increased but its activity decreased (43 % and 43 % respectively, p < 0.05 vs control). Total degradative capacity of kidney tissue from diabetic rats was also lower (Control: 48 ± 7 %, Diabetic: 33 ± 6 %, p < 0.05). Activation of latent MMPs with amino-phenylmercuric acetate increased matrix degradation by two-fold. However the relative decrease associated with experimental diabetes still remained. All diabetes-associated changes in MMP and TIMP mRNA and activities were attenuated by perindopril treatment in association with reduced type IV collagen accumulation. Conclusions/interpretation: These results indicate that the impairment of matrix degradation contributes to matrix accumulation in diabetic nephropathy and that the beneficial effects of ACE inhibition could in part be mediated by modulation of changes in matrix degradative pathways. [Diabetologia (2002) 45: 268–275] Received: 13 June 2001 and in revised form: 14 September 2001     

Islet transplants in diabetic Lewis rats prevent and reverse diabetes-induced increases in vascular permeability and prevent but do not reverse collagen solubility changes

Summary The effects of islet transplantation on diabetes-induced increases in vascular permeability and collagen solubility were examined in new granulation tissue vessels and collagen formed after induction of streptozotocin diabetes in male Lewis rats. Albumin permeation was increased by 50% (p<0.001) and collagen solubility was decreased by 50% (p<0.001) in granulation tissue from untreated diabetic animals as compared with controls. The islet transplants reversed diabetes-induced vascular permeability increases in tissues formed prior to islet transplantation (tissue to blood isotope ratio =2.1±0.1 - SD for controls, 3.2±0.2 for diabetic rats and 2.0±0.2 for diabetic rats given islets) and prevented permeability increases in new tissues formed following transplantation (tissue to blood isotope ratio =2.1±0.1 for controls, 3.3±0.8 for diabetic rats and 1.9±0.2 for diabetic rats given islets). In contrast, while islet transplants prevented diabetes-induced decreased collagen solubility in tissues formed after transplantation (controls = 24%, diabetic rats =12%, and diabetic rats given islets =24%), collagen solubility in tissues formed prior to islet transplantation was virtually unaffected. These findings indicate that collagen changes induced by the diabetic milieu are not nearly as readily reversed by normalization of the diabetic milieu as (diabetes-induced) alterations in vascular functional integrity.     

Aortic atherosclerosis in diabetes mellitus is associated with an insertion/deletion polymorphism in the angiotensin I-converting enzyme gene

Summary An insertion(I)/deletion(D) polymorphism in the angiotensin I-converting enzyme (ACE) gene seems to be associated with clinical heart disease in patients with diabetes mellitus. It is not known whether increased atherosclerosis or other factors among individuals with certain ACE-gene subtypes form the basis for the increased prevalence of heart disease among these subjects. We measured, at autopsy, the extent of macroscopically visible aortic atherosclerosis in 22 diabetic and 39 non-diabetic subjects and determined the ACE-genotype of all individuals by the polymerase chain reaction. The percentage of aortic surface area covered with atherosclerotic lesions was 29±8 (n=6), 71±7 (n=9), and 65±7 (n=5) in the II-, ID-, and DD-genotype subgroups, respectively, among diabetes patients (mean ± SEM) (2 p<0.01, when comparing values from the ID and DD groups to the II group). The values were 37±9 (n=11), 40±5 (n=14) and 37±6 (n=11) in the II-, ID-, and DD-genotypes in the non-diabetic group. There were no differences in sex ratio or age in any of the ACE-gene subtypes. The previously described relationship between heart disease and the ACE-gene polymorphism in diabetes could thus be founded in an increased extent of atherosclerosis among patients with the ID- and DD-ACE-gene subtypes. Patients with diabetes have several alterations in the composition of the collagenous components in the arterial wall. We also analysed for associations between total collagen and type IV and type V collagen content in the aortic vessel wall and the ACE-gene subtypes. We were, however, not able to disclose correlations between the polymorphism and any of these parameters. In conclusion, our data show an association between the ACE-I/D polymorphism and the degree of aortic atherosclerosis in diabetes; however, we did not observe correlations between the polymorphism and data concerning arterial collagenous components.Abbreviations ACE Angiotensin converting enzyme - PCR polymerase chain reaction - IDDM insulin-dependent diabetes mellitus - NIDDM non-insulin-dependent diabetes mellitus     

Suppression of transforming growth factor beta and vascular endothelial growth factor in diabetic nephropathy in rats by a novel advanced glycation end product inhibitor, OPB-9195

Aims/hypothesis. Advanced glycation end products (AGEs) participate in the pathogenesis of diabetic nephropathy. We reported earlier that OPB-9195, a synthetic thiazolidine derivative and novel inhibitor of advanced glycation, prevented progression of diabetic glomerulosclerosis by lowering serum concentrations of advanced glycation end products and reducing their deposition in the glomeruli. Here, we examined their contribution and that of growth factors, such as transforming growth factor-β (TGF-β) and vascular endothelial growth factor (VEGF), to the progression of diabetic nephropathy. We also investigated the expression of type IV collagen in the kidneys of Otsuka-Long-Evans-Tokushima-Fatty (OLETF) rats, a Type II (non-insulin-dependent) diabetes mellitus model, after treatment with OPB-9195. Methods. Using northern blots and immunohistochemical techniques, we determined the renal expression of TGF-β and type IV collagen mRNAs and proteins in OLETF rats. We also examined OPB-9195's effects on renal expression of VEGF mRNA and protein. Results. Concomitant increases in TGF-β and type IV collagen expression were observed at each point in time in OLETF rats not given OPB-9195. In contrast, OPB-9195 treatment greatly suppressed the renal expression of TGF-β, VEGF and type IV collagen mRNAs and proteins to that seen in non-diabetic rats. Conclusion/interpretation. Since OPB-9195, an AGE-inhibitor, prevented the progression of diabetic nephropathy by blocking type IV collagen production and suppressing overproduction of two growth factors, TGF-β and VEGF, in diabetic rats, this compound warrants further investigation. [Diabetologia (1999) 42: 579–588] Received: 2 October 1998 and in final revised form: 28 December 1998     

Biochemical markers of types I and III collagen and limited joint mobility in type 1 diabetic patients

Abstract. Limited joint mobility (LJM), a long-term complication of diabetes, has been shown to be associated with microvascular complications of diabetes. Connective tissue alterations may contribute to the development of LJM and other diabetic complications. We tested whether biochemical markers of types I and III collagen metabolism are associated with LJM in type 1 diabetes. We studied 28 male patients of mean age 43.4 years (SD=9.5) and with a duration of diabetes of 25.2 years (SD=9.7) years. LJM assessment included goniometric measurements of the joints and classification by Rosenblooms method. We measured serum concentrations of aminoterminal propeptide of type III procollagen (PIIINP), carboxyterminal propeptide of type I procollagen (PICP) and carboxyterminal crosslinked telopeptide of type I collagen (ICTP); urinary excretion of crosslinked N-telopeptides of type I collagen (NTX) and deoxypyridinoline crosslinks (DPyr) was also measured. Although average serum PIIINP tended to be higher in subjects with moderatesevere LJM (3.1±1.3 µg/l) than in subjects with mild LJM (2.5±0.7 µg/l) or without LJM (2.6±0.4 µg/l), no significant association was found (p<0.27). Concentrations of the other collagen markers were not different in subjects with or without LJM. We conclude that synthesis and degradation of types I and III collagen in diabetic subjects with LJM did not differ from those without LJM to reflect changes in the biochemical markers of these proteins.     

Ultrastructural alterations in capillaries of the diabetic hypertensive rat retina: protective effects of ACE inhibition

Aims/hypothesis The ACE inhibitor cilazapril was administered to diabetic hypertensive rats to evaluate its ability to influence the development of retinal capillary alterations.Methods Normotensive (strain: Wistar Kyoto) and genetically hypertensive (strain: spontaneously hypertensive) rats were rendered diabetic by intravenous injections of streptozotocin. Half of the diabetic animals received cilazapril with their daily food. At 20 weeks of diabetes, endothelial cells, pericytes and extracellular matrix were assessed by ultrastructural morphometry. Each experimental group consisted of seven animals.Results Cilazapril normalised systolic arterial pressure in diabetic hypertensive rats (137±2 mm Hg compared with 188±16 mm Hg in non-medicated diabetic hypertensive rats, p<0.001). The number of endothelial intercellular junctions was reduced in untreated diabetic hypertensive rats (0.15±0.05, p<0.02, vs 0.47±0.20 in non-diabetic normotensive rats). In diabetic hypertensive animals treated with cilazapril, this loss was attenuated (0.32±0.16, p<0.05). The significant thickening of the basement membrane observed in the diabetic normotensive (132.8±19.4 nm) and diabetic hypertensive (150.3±20.2 nm) groups was decreased by cilazapril in the diabetic hypertensive group (116.7±11.0 nm, p<0.01), but was unaffected in the normotensive (131.9±17.3 nm) group. No protective effect of the drug was observed in either group on pericytes.Conclusions/interpretation Long-term administration of an effective antihypertensive therapy normalises endothelial alterations and basement membrane thickness in diabetic hypertensive conditions, and thus may account for the well-known improvement of the blood-retinal barrier observed during antihypertensive treatment.Abbreviations ANGII angiotensin II - RAS renin-angiotensin system - SHR spontaneously hypertensive rat - STZ streptozotocin - VEGF vascular endothelial growth factor - WKY Wistar Kyoto     

The presence of genetic hypertension stimulates early renal accumulation of fibronectin in experimental diabetes mellitus

Aims/hypothesis: To investigate the interaction between hypertension and diabetic nephropathy, we studied the renal accumulation of fibronectin in a genetic model of hypertension with streptozotocin-induced diabetes mellitus. Methods: Spontaneously hypertensive rats (SHR) and their genetically normotensive control Wistar Kyoto rats (WKY) were studied at 4 weeks of age. The rats were killed 20 days after the induction of diabetes mellitus. The renal accumulation of fibronectin was estimated using Western blot analysis as well as immunofluorescence technique and confocal microscopy. Results: Blood glucose concentrations were similar in diabetic SHR rats (27 ± 3.3 mmol/l) and WKY rats (25.5 ± 2.7 mmol/l). The systolic blood pressure was higher in both groups of SHR rats than in the control rats. The abundance of renal fibronectin as detected by Western blot analysis was (p < 0.05) higher in the diabetic SHR rats (41.4 ± 15.0 densitometric U, n = 8) than in the control rats, and no difference was observed between diabetic and control WKY rats (20.8 ± 6.2, n = 8) and (27.8 ± 17.2, n = 8). The mean peak intensity of fibronectin signal within the glomerulus as estimated by confocal microscopy was higher (p < 0.05) in the diabetic SHR rats (32.9 ± 3.5) than in control SHR rats (11.9 ± 5.7) or diabetic (7.4 ± 2.2) and control (15.2 ± 7.9) WKY rats. Conclusion/interpretation: In experimental diabetes the presence of genetic hypertension promotes earlier accumulation of renal fibronectin, a matrix protein implicated in renal glomerulosclerosis. [Diabetologia (2001) 44: 2088–2091] Received: 11 January 2001 and in revised form: 30 May 2001     

Kidney tissue somatomedin C and initial renal growth in diabetic and uninephrectomised rats

Summary Kidney growth after induction of experimental diabetes in rats was compared to compensatory renal growth in response to unilateral nephrectomy. After 4 days of diabetes, kidney weight had increased from 816±21 mg (SEM) to 940±42 mg (15%). In insulin-treated diabetic rats kidney weight was unchanged at the end of the study, namely 828±15 mg.In unilaterally nephrectomised rats kidney weight increased from 840±20 mg (SEM) to 1050±60 mg during 4 days (24%). We observed increased kidney content of somatomedin C in both diabetic and uninephrectomised rats. In untreated diabetic rats it was maximal after 48 h, with an increase of 77% (3469±312 ng/g (SEM) versus 1961±173 ng/g). After 4 days the somatomedin C content had returned to initial levels. In insulin-treated rats somatomedin C content did not increase during the observation period. The somatomedin C content of the remaining kidney after unilateral nephrectomy was maximal after 24 h with an increase of 58% (from 1340±203 ng/g (SEM) to 2122±214 ng/g). The somatomedin C content returned to normal at day 4. Serum somatomedin C declined insignificantly in diabetic animals during the experimental period, but a significant decrease (p<0.02) was found in uninephrectomised rats. This study demonstrates that kidney somatomedin C peaks during the first or second day after uninephrectomy or induction of diabetes, respectively, and that insulin treatment sufficient to prevent kidney growth abolishes the increase. These similar rapid initial hypertrophies/hyperplasies may thus be dependent on local somatomedin C formation.     

Impaired arteriolar mechanotransduction in experimental diabetes mellitus

Decreased arteriolar distensibility in diabetes may impair signal transduction mechanisms that are required for converting a pressure stimulus into smooth muscle contraction. These studies aimed to determine if pressure-induced increases in arteriolar intracellular Ca(2+) are altered in diabetes and whether diabetes is associated with alterations in composition of the extracellular matrix. Studies of mechanical properties used single, isolated, and cannulated cremaster arterioles from streptozotocin (60 mg/kg) diabetic rats and age-matched controls. To measure Ca(2+)(i), arterioles were loaded with Fura 2 (5 microM) after which preparations were examined by fluorescence microscopy and image analysis. Matrix protein (type IV collagen, laminin, fibronectin) deposition was studied by immunohistochemistry. Over a range of 30-120 mm Hg control vessels showed a linear relationship (r = 0.98, p < 0.01) between intraluminal pressure and Ca(2+)(i). Vessels from diabetic animals also showed a linear relationship (r = 0.99, p < 0.01), however, the mean slope was significantly (p < 0.02) less in the diabetic (0.17 +/- 0.05, n = 5) compared to controls (0.51 +/- 0.09, n = 7). Similarly, the slope of the wall tension-Ca(2+)(i) relationship was significantly decreased in vessels from diabetic animals. These differences were ameliorated by treatment of diabetic animals (n = 5) with aminoguanidine. Increased content of type IV collagen, laminin and fibronectin in vessel media was evident after 2 weeks of diabetes and showed a further increase with duration of diabetes. The data suggest that for a given increase in luminal pressure arterioles from diabetic animals response with an attenuated rise in smooth muscle Ca(2+)(i). This mechanotransduction defect may relate to alterations in the composition of the extracellular matrix within the arteriolar wall.     

Aminoguanidine inhibits the development of accelerated diabetic retinopathy in the spontaneous hypertensive rat

Summary Arterial hypertension has been identified as a major secondary risk factor for diabetic retinopathy. However, the mechanisms by which hypertension worsens retinopathy are unknown. Inhibition of advanced glycation product formation prevents the development of experimental diabetic retinopathy in normotensive diabetic rats. In this study the effect of hypertension on the rate of diabetic retinopathy development and the formation of arteriolar thrombosis was evaluated. We also evaluated the effect of aminoguanidine, an inhibitor of advanced glycation end product formation on retinal pathology of diabetic hypertensive rats. After 26 weeks of diabetes, hypertension accelerated the development of retinopathy despite a lower mean blood glucose level than in the non-hypertensive group (diabetic spontaneous hypertensive rats (SHR) 16.00±6.83 mmol/l; diabetic normotensive Wistar Kyoto rats (WKY) 34.9±3.64 mmol/l; p<0.0001). Diabetic SHR had nearly twice as many acellular capillaries as diabetic WKY (SHR diabetic: 91.9±7.5 acellular capillaries per mm² of retinal area vs WKY diabetic: 53.7±8.5 acellular capillaries per mm² of retinal area), and a 3.8-fold increase in the number of arteriolar microthromboses (SHR diabetic 23504±5523 m² vs SHR non-diabetic 6228±2707 m²). Aminoguanidine treatment of SHR diabetic rats reduced the number of acellular capillaries by 50%, and completely prevented both arteriolar deposition of PAS-positive material and abnormal microthrombus formation. These data suggest that hypertension-induced deposition of glycated proteins in the retinal vasculature plays a central role in the acceleration of diabetic retinopathy by hypertension.     

Extracellular matrix in human diabetic nephropathy: reduced expression of heparan sulphate in skin basement membrane

Summary In diabetic nephropathy, expression of glycosaminoglycan side chains of heparan sulphate proteoglycan in the glomerular basement membrane is reduced proportionally to the degree of proteinuria. We performed a cross-sectional study to evaluate whether non-vascular basement membranes also show a decrease in heparan sulphate side chain staining in patients with diabetic nephropathy. We evaluated the skin basement membrane for extracellular matrix components in the following groups: control subjects (n = 16); patients with Type 1 diabetes and normoalbuminuria (n = 17), microalbuminuria (n = 7), and macroalbuminuria (n = 16); patients with Type 1 diabetes and diabetic nephropathy undergoing renal replacement therapy (n = 13); and non-diabetic patients undergoing renal replacement therapy (n = 21). The following antibodies were used for this immunohistochemical study: monoclonal antibodies against the heparan sulphate side chain (JM403) and core protein (JM72) of the glomerular heparan sulphate proteoglycan; polyclonal antibodies against the core protein (B31); polyclonal antibodies against collagen types I, III, and IV, fibronectin, and laminin; and monoclonal antibodies against the non-collagenous domain of α1(collagen IV) and α3(collagen IV), against transforming growth factor β(2G7), and against advanced glycosylation end products (4G9). Expression of heparan sulphate side chains was reduced in the skin basement membrane of patients with overt diabetic nephropathy, of those with Type 1 diabetes undergoing renal replacement therapy, and those with non-diabetic renal failure. Increased intensity of staining was found for collagen type I and advanced glycosylation end products in patients with diabetic nephropathy. Changes in the extracellular matrix of the skin basement membrane seem to be similar to those in the glomerular basement membrane. These findings support the suggestion that patients with diabetic nephropathy also have altered heparan sulphate and collagen staining in extrarenal basement membranes. However, patients with non-diabetic renal failure also had reduced expression of heparan sulphate in the skin basement membrane, suggesting that this finding is not specific for diabetic nephropathy. [Diabetologia (1998) 41: 791–798] Received: 13 November 1997 and in revised form: 10 February 1998     

Strict insulin therapy normalises organ nitrogen contents and the capacity of urea nitrogen synthesis in experimental diabetes in rats

Summary Rats with experimental diabetes due to streptozotocin (75 mg/kg body weight) and free access to food were divided into two groups. One group (n=9) was optimally treated with insulin (glucosuria <4.0 mmol/24 h), using heat treated very long-acting ultralente insulin. The other group (n=10) was poorly treated with insulin (glucosuria 20–30 mmol/24 h).The nitrogen balance and energy balance of optimally treated diabetic rats was positive and not different from the control group (n=6). In the poorly treated diabetic rats the nitrogen balance was reduced whereas the energy balance was not different from that of control rats. After 4 weeks the fasting glucagon was: 50±21 ng/l (mean±SEM) in control rats, 62±18 ng/l in optimally treated diabetic rats and 249±58 ng/l in poorly treated diabetic rats (p<0.01). The capacity of urea nitrogen synthesis determined during alanine loading was: 9.6±1.0 umol/(min 100 g body weight) in control rats, 10.6±1.7 umol/(min 100 g body weight) in optimally treated diabetic rats and 17.3±1.3 umol/(min 100 g body weight) in poorly treated diabetic rats (p<0.01).Nitrogen contents of carcass, heart, intestines, liver, and kidneys as determined by Kjeldahl analyses were identical in control rats and optimally treated diabetic rats. In the poorly treated diabetic rats carcass-nitrogen and heart-nitrogen contents were reduced to 89% of the control value (p<0.01), whereas the kidney-nitrogen content was increased to 112% of the control value (p<0.01).Strict insulin therapy in experimental diabetes leads to a normalisation of nitrogen metabolism and hyperglucagonaemia, whereas less than optimally insulin treated rats show marked abnormalities in nitrogen metabolism as well as hyperglucagonaemia.